Hydroxy,acyloxy and 11-keto-1,3,5(10),7-estratetraenes



United States Patent 3,517,036 HYDROXY, ACYLOXY AND ll-KETO-1,3,5(),7-ESTRATETRAENES Patrick A. Diassi, Westfield, N.J., assignor toE. R. Squibb & Sons, Inc., New York, N.Y., a corporation of Delaware NoDrawing. Filed Nov. 15, 1966, Ser. No. 594,372 Int. Cl. C07c 169/10 U.S.Cl. 260397.3 6 Claims ABSTRACT OF THE DISCLOSURE Hydroxy, acyloxy and11-keto-1,3,4(l0),7-estratetranes are prepared from 19-nor-A-androstadienes by a two-step process, entailing initially thehydroxylation or ketonization of the androstadiene to yield hydroxy orll-keto 19-nor-A -androstadiene intermediates, which are new compounds,and then the l-dehydrogenation of these new intermediates to yield thefinal products. The estratetraenes are antigonadotropic agents.

This invention relates to and has for its object the provision of newhydroxy-7-dehydroestranes (i.e., hydroxy 1,3,5 (10),7 estratetraenes),acyloXy-7-dehydroestranes and 1l-keto-7-dehydroestranes, processes forpreparing the same, and new intermediates useful in said processes.

It has been found that a l9'-nor-A -androstadiene may be converted to ahydroxy-7-dehydroestrane or ll-keto- 7-dehydroestrane derivative in highyield by a two-step process without any substantial formation ofundesired by-produc-ts. In essence, therefore, the process of thisinvention entails subjecting a 19-nor-A -androstadiene to the action ofenzymes of a hydroxylating or ketonizing microorganism, whereby acorresponding hydroxy-19- nor A -androstadiene or 1l-keto-l9-nor-A-androstadiene derivative is formed; and subjecting the latter to theaction of enzymes of a l-dehydrogenating microorganism, to yield thedesired hydroxy-7-dehydroestrane or 11-keto-7-dehydroestrane finalproduct.

Among the suitable starting steroids are included any of the l9-norA-androstadienes. The preferred starting steroids, however, are the3,17-dioXygenated-19-nor-A androstadienes, such as l9-nor-A-androstadiene-3,17- dione, 19-nor-7-dehydrotestosterone,19-nor-l7u-methyl- 7-dehydrotestosterone and 19'-nor-17u-ethyny1 7dehydrotestosterone.

In the first step of the process of this invention, the steroidsubstrate is subjected to the action of enzymes of a hydroxylating orketonizing microorganism, the reaction being carried out in the usualmanner by culturing the microorganism in the presence of the steroid, orby treating the steroid with non-proliferating cells of themicroorganism, or by intermixing the steroid with bydroxylating orketonizing enzymes previously obtained from the microorganism. Theconditions for such microbial reaction are well known in the art and aresimilar to those specified in U.S. Pat. 3,179,698.

Any known hydroxylating microorganism can be used as the source of thehydroxylating enzyme. Such microorganisms include, inter alia,llB-hydroxylating microorganisms, such as Curvularz'a helleb ori andCunninghamella blakesleeana; lla-hydroxylating microorganisms, such asFusarz'um javanicum var. ensifo rme, Aspergitllus ochraceus, Rhizopusnigricans and Aspergillus nidwlans; l6a-hydroxylating microorganism,such as Streptomyces roseochromogenes, Hypomyces aurantius, Pestalotia:funerea, Streptomyces viridis, Streptomyces o livaceus and Streptomycescalifornicus; '6-hydroxylating microorganisms, such as Tricothecium'roseu'm, Cunninghamella 3,517,036 Patented June 23, 1970 ice elegans,Rhizopus arrhizus, Syncepha lastrum racemosum and Circinella linderi;2a-hydroxylating microorganisms, such as Streptomyces roseochromogenesand Nocardia italica; l4a-hydroxylating microorganisms, such asCaldariomyces fumago, Helminthosporium buchloes, Bacillus cereus andMu'cor griseocyanus; l5a-hydroxylating microorganisms, such asColletotrium linicola, Penicillium sp. (ATCCll, 598) and Fusariumorthoceras; and IZfi-hydroxylating microorganisms, such as Diplodianatalensis, Calonectria decora: and Colletotrichum derria'is.

The first step in the process results in the preparation of the newhydroxy-19-nor-A -androstadiene intermediates of this invention. Thepreferred intermediates are the hydroxy 3,l7-dioxygenated-l9-nor-A-androstadienes, particularly those of the formula wherein one X ishydroxy and the remaining Xs are hydrogen, or where two Xs are connectedto the same carbon, these two Xs can represent oxo (0 and Y is hydroxy,Z is hydrogen, methyl or ethynyl, or together Y and Z is 0x0. Thell-keto compounds can be prepared from the corresponding ll-hydroxycompounds by oxidizing the latter, as by treatment with chromic oxide,or directly by treating a 19-nor-A -androstadiene with the enzymes of anll-ketonizing microorganism, such as Corticium microsclerotia.

These hydroxy-19-nor-A -"-androstadienes or ll-ketol9-nor-A-androstadienes are then subjected to the action of enzymes of al-dehydrogenating microorganism, to yield the desiredhydroxy-7-dehydroestrane or ll-keto- 7-dehydroestrane final products,the reaction being carried out in the usual manner by culturing themicroorganism in the presence of the steroid, or by treating the steroidwith non-proliferating cells of the microorganism, or by intermixing thesteroid with l-dehydrogenating enzymes previously obtained from themicroorganisms. Optimally, the dehydrogenation is conducted withcellfree extracts of l-dehydrogenating microorganisms, as by the methodand with the enzymes described in U.S. Pat. 3,047,469.

The second step in the process results in the formation of certain ofthe final products, namely the hydroxy-7- dehydroestrane and1l-keto-7-dehydroestrane derivatives, and preferably thehydroxy-3-l7-dioxygenated-7-dehydroestranes and1l-keto-3,17-dioxygenated-7-dehydroestranes. The hydroxy final productmay be esterified by treating with an acyl halide or acid anhydride inthe presence of a base, such as pyridine, to yield the correspondingesters where the 3- and other hydroxy groups are esterified. Thepreferred acylating agents are the acyl chlorides or acid anhydrides ofhydrocarbon carboxylic acids of less than tweleve carbon atoms, asexemplified by the lower aklanoic acids (e.g., acetic, propionic, andhexanoic acid), the lower alkenoic acids, the monocyclic aryl carboxylicacids (e.g., benzoic acid), the monocyclic aryl lower alkanoic acids(e.g., phenylacetic acid and ,S-phenylpropionic acid), the lowercycloalkane carboxylic acid, and the lower cycloalkene carboxylic acids.Those of the final products that contain a hydroxy group on a secondarycarbon atom can also be oxidized, as by treatment with chromic oxide, toyield the corresponding keto derivatives.

Rz Y

wherein Y and Z are as hereinbefore defined, one R is R'O and theremaining Rs are hydrogen, or where two Rs are connected to the samecarbon atom, these tw Rs can represent x0 (0 and R is hydrogen, or acyl(particularly the acyl radical of a hydrocarbon carboxylic acid of lessthan twelve carbon atoms).

The final products of this invention are useful as antigonadotropicagents, for which purpose they are administered in the same manner asknown estrogens, such as estradiol valerate, in the treatment ofsymptoms of menopause, etc. They are especially well suited for thispurpose since they do not possess any appreciable amount of estrogenic,anti-estrogenic and hypocholesteremic activities.

The following examples illustrate the invention (all temperatures beingin centigrade):

EXAMPLE I 11a-hydroxy-19-nor-A -androstadiene-3,17-dione (A)Fermentati0n..Surface growth from each of two 10-day old agar slantcultures of Fusarium javanicum var. ensiforme (QM-524) (ArmyQuartermaster, Natick, Mass), the slant containing as a nutrient medium(A):

Grams Glucose 10 Yeast extract 2.5

K HPO 1 Agar 20 Distilled water to 1 liter.

is suspended in 5 ml. of a 0.01% sodium lauryl sufate aqueous solution.One ml. portions of the suspension are used to inoculate five 250 ml.concial flasks, each containing 50 ml. of the following sterilizedmedium (B):

Grams Dextrose Corn steep liquor 6 NH H PO 3 Yeast extract 2.5

CaCO 2.5

Distilled water to 1 liter.

After 72 hours of incubation at 25 with continuous rotary agitation (280cycles/minute, 2 inch stroke) 10% 10% (vol/vol.) transfers are made toten 250 ml. conical flasks each containing 50 ml. of fresh sterilizedmedium B plus 3 milligrams ml. of 19 nor A androstadiene-3,17-dione. Thesteroid is added by supplementing each flask with 0.50 ml. of a sterilesolution of the steroid in N,N-dirnethylformamide containing 300 mg./ml.of steroid. A total of 1.5 grams is used. After 7 days of furtherincubation, the contents of the flasks are pooled through a Seitzclarifying pad. The flasks, mycelium and pads are washed with successive50 ml. portions of warm water. The combined filtrate and washings have avolume of 500 ml.

(B) Is0Iati0n.The combined filtrate and washings (500 ml.) are extractedthree times with 160 m1. portions of chloroform. The combined chloroformextracts are washed with water, dried over anhydrous sodium sulfate andconcentrated to dryness under vacuum, leaving about 520 mg. of crudeproduct. This material is chromatographed on a thin layer of Silica GelGF (Merck) with chloroform containing 5% (by volume) methanol as thedeveloping solvent. The major u.v.-absorbing band is eluted with a 1:1(by volume) mixture of methanol and chloroform. After evaporating offthe solvent the residue is partitioned between chloroform and a 1:1 (byvolume) mixture of water and methanol. The choloform phase, uponevaporation under vacuum to dryness, yields crystalline1la-hydroxy-19nor-A -androstadiene-3, 17-dione.

EXAMPLE 2 11ot-hydroxy-7-dehydro-19-nortestosterone Following theprocedure of Example 1, but substituting an equivalent amount of7-dehydro-19-nortestosterone for the 19-nor-A -androstadiene-3,17-dioneand Aspergillus oclzraceus (NRRL-405) for the Fusarium javanicum, the11m hydroxy 7-dehydro-l9-nortestosterone crystallizes directly from thechloroform extract upon evaporation without purification by the thinlayer chromatography.

Similarly, by following the procedure of Example 1, but substituting thefollowing microorganisms for the Fusarium used in the example,11a-hydroxy-19-nor-A androstadiene-3,17-dione is Obtained: Rhizopusnigricans ATCC 15441, and Aspergillus nidulans ATCC 11267.

Moreover, by substituting the following steroid substrates for the19-nor-A -androstadiene-3,17-dione in the procedure of Example 1, theindicated product is obtained:

Steroid substrate Product 19-uor-17a-methyl-7-dehydrotestosterone.dehydrotestosterone.

19-nor-17uethynyl-7-dehydro- 11a-hydroxy-19-nor17a-ethynyl-7testosterone. dehydrotestosterone.

EXAMPLE 3 1IB-hydroxy-l9-nor-A -androstadiene-3,17-dione EXAMPLE 46fl-hydroxy-19-nor-A -androstadiene-3, l7-dione Following the procedureof Example 1, but substituting T ricotlzecium roseum (ATCC12519) for themicroorganism used in Example 1, 6fl-hydroxy-19-nonAandrostadiene-3,17-dione is obtained.

EXAMPLE 5 19-nor-A -androstadiene-3,11,17-trione (A)Fermentation.-Surface growth from each of two IO-day old agar slantcultures of Corticium microsclerotia (NRRL-2727), the slant containingas a nutrient medium (A):

Grams Oatmeal 20 Tomato paste 20 Agar 15 Distilled water to 1 liter.

is suspended in 6 ml. of a 0.01% sodium lauryl sulphate aqueoussolution. Three ml. portions of the suspension are used to inoculatefour 250 'ml. conical flasks, each containing 50 ml. of the followingsterilized medium (B):

Grams Glucose 30 Soy bean meal 20 Soy bean oil 2.2 CaCO 2.5

Distilled water to 1 liter.

5 After 72 hours of incubation at 25 with continuous rotary agitation(280 cycles/minute; 2 inch stroke) 10% (vol/vol.) transfers are made totwenty 250 ml. conical flasks, each containing 50 ml. of the followingsterllized medium (C):

Grams Dextrose 10 Corn steep liquor 6 NH H PO 3 Yeast extract 2.5 CaCO2.5

Distilled water to 1 liter.

After 24 hours of incubation, using the same conditions as describedabove, the steroid (500 micrograms/ ml.) is added by supplementing eachflask with 0.25 ml. of a sterile solution (100 mg./ml.) of 19-nor-Aandrostadiene-3,17-dione in N,Ndimethylformamide. A total of 500 mg. isfermented. After approximately 10 hours of further incubation usingidentical conditions as described above, the fermentation is harvested.The contents of the flasks are pooled; the broth has a volume of 1,000ml.

(B) Islati0n.The broth is extracted three times with 200 ml. portions ofchloroform. The combined chloroform extracts are washed with water,dried over anhydrous sodium sulfate and concentrated to dryness undervacuum, leaving about 150 mg.. of crude product. This material ischromatographed on a thin layer of Silica Gel GF (Merck) with chloroformcontaining 5% (by volume) methanol as the developing solvent. Theu.v.-absorbing band which moves with the mobility of the substrate,19-norandrostenedione, is eluted with a 1:1 (by volume) mixture ofmethanol and chloroform. After evaporating off the solvent the residueis partitioned between chloroform and a 1:1 (by volume) mixture of waterand methanol. The chloroform phase, upon evaporation under vacuum todryness, yields crystalline 11-keto-19- nor-A"-androstadiene-3,17-dione.

EXAMPLE 6 2a-hydroxy-19-nor-A -androstadiene-3, l7-dione (A)Fermentati0n.-Surface growth from each of two 10-day old agar slantcultures of Streptomyces roseochromogerzes (ATCC 13400), the slantcontaining as a nutrient medium (A):

Grams Glucose 1O Yeast extract 2.5 K HPO 1 Agar 20 Distilled water to 1liter.

is suspended in 5 ml. of a 0.01% sodium lauryl sulphate aqueoussolution. One ml. portion of the suspension is used to inoculate eight250 ml. conical flasks, each containing 50 ml. of the followingsterilized medium (B):

Grams Glucose 30 Soy bean meal 20 Soy bean oil 2.2 CaCO 2.5

Distilled water to 1 liter.

After 72 hours of incubation at 25 with continuous rotary agitation (280cycles/minutes, 2 inch stroke) 10% (vol/vol.) transfers are made to onehundred 250 ml. conical flasks, each containing 50 ml. of freshlysterilized medium B. After 24 hours of further incubation, using thesame conditions as described above, the steroid (300 micrograms/ml.) isadded by supplementing each flask with 0.25 ml. of a sterile solution(60 mg./ml.) of l9-nor-A '7-androstadiene 3,17 dione inN,N-dimethylformamide. A total of 1.5 g. is fermented. Afterapproximately 78 hours of further incubation, using identical conditionsas described above, the fermentation is harvested The contents of theflasks are pooled through a Seitz clarifying pad. The flasks, myceliumand pads are washed with successive ml. portions of warm water. Thecombined filtrate and washings have a volume of 5 500 ml.

(B) Isolation and characterizati0n.-The combined filtrate and washings(5500 ml.) are extracted three times with 1500 ml. portions ofchloroform. The combined chloroform extracts are washed with water,dried over anhydrous sodium sulfate and concentrated to dryness undervacuum, leaving about 900 mg. of crude product. This material ischromatographed on a thin layer of Silica Gel GF (Merck) with chloroformcontaining 5% (by volume) methanol as the developing solvent. Theu.v.-absorbing band which moves with the mobility of the substrate,l9-norandrostenedione, is eluted with a 1:1 (by volume) mixture ofmethanol and chloroform. After evaporating off the solvent, the residueis partitioned between chloroform and 1:1 (by volume) mixture of waterand methanol. The chloroform phase, upon evaporation under vacuum todryness, yields crystalline 20chydroxy-19nor-A -androstadiene-3, 17-dione.

EXAMPLE 7 Following the procedure of Example 6, but substitutingNocardia italica for the Streptamyces roseochromogenes 2oz hydroxy 19nor A androstadiene 3,17 dione is obtained.

Similarly, by substituting the following steroid substrates for the19=nor-A -androstadiene-3,17-dione in the procedure of Example 6, theindicated product is obtained:

Steroid substrate Product 19-nor-17a-methy1-7-dehydrotestos-2a-hydroxy-l9-nor-17a-methyl-7- terone. dehydrotestosterone.

terone. dehydrotestosteroue. 1llnor-7-dehydrotestosterone 2ahydroxy-19nor-7-dehydrotestosteroue.

EXAMPLE 8 16a-hydroxy-19-nor-A -androstadiene-3,17-dione (A)Fermentatiom-Surface growth from each of 2 10-day old agar slantcultures of Streptomyces roseochromogenes (ATCC 13400), the slantcontaining as a nutrient medium (A):

Grams Glucose 10 Yeast extract 2.5 K HPO 1 Agar 20 Distilled water to 1liter.

is suspended in 5 ml. of a 0.01% sodium lauryl sulfate aqueous solution.One ml. portions of the suspension are used to inoculate eight 250 ml.conical flasks, each containing 50 ml. of the following sterilizedmedium (B):

Grams Glucose 30 Soy bean meal 20 Soy bean oil 2.2

CaCO 2.5 Distilled water to 1 liter.

hours of further incubation, using identical conditions as describedabove, the fermentation is harvested. The contents of the flasks arepooled through a Seizt clarifying pad. The flasks, mycelium and pads arewashed with successive 50 ml. portions of warm water. The combinedfiltrate and washings have a volume of 2,000 ml.

(B) Isolation and characterization.--The combined filtrate and washings(2,000 ml.) are extracted three times with 500 ml. portions ofchloroform. The combined chloroform extracts are washed with water,dried over anhydrous sodium sulfate and concentrated to dryness undervacuum, leaving about 300 mg. of crude product. This material ischromatographed on a thin layer of Silica Gel GF (Merck) with chloroformcontaining (by volume) methanol as the developing solvent. The majoru.v.-absorbing band is eluted with a 1:1 (by volume) mixture of methanoland chloroform. After evaporating oflf the solvent, the residue ispartitioned between chloroform and 1:1 (by volume) mixture of water andmethanol. The chloroform phase, upon evaporation under vacuum todryness, yields crystalline 16a-hydroxy-19-nor- A-androstadiene-3,17-dione.

EXAMPLE 9 EXAMPLE 14a-hydroxy-19-nor-A -androstadiene-3,17-dioneFollowing the procedure of Example 1, but substituting Caldariomycesfumago (ATCC-11925) for the microorganism used in the example,14a-hydroxy-19-nor-A -androstadiene-3,l7-dione is obtained.

EXAMPLE 1 1 ot-hydroxy-19-nor-A "-andr0stadiene-3,17-dione Following theprocedure of Example 1, but substituting Colletolrichum linicola(NCTC-l194) for the microorganism used in Example 1,15ot-hydroxy-19-nor-A -androstadiene-3,l7-dione is obtained.

EXAMPLE 12 IZfi-hydroxy-19-nor-A -androstadiene-3,17-dione Following theprocedure of Example 1, but substituting Diplodia natalensis (ATCC-9055)for the microorganism used in the example, IZB-hydroxy-19-rlor-A-androstadiene-3,17-dione is obtained.

EXAMPLE 13 1 1a-hydroxy-7-dehydroestrone (A) Fermentati0n.Surface growthfrom a two-week old agar slant of Corynebacterium simplex (ATCC 6946),the slants containing as a nutrient medium (A):

Grams Glucose 10 Yeast extract 2.5 K HPO 1 Agar 2 0 Distilled water to 1liter.

is suspended in 5 ml. of 0.01% aqueous sodium lauryl sul fate solution.One ml. portions of this suspension are used to inoculate four 250Erlenmeyer flasks, each containing 50 ml. of the following sterilizedmedium (B):

Grams Beef extract 1.5

Yeast extract 3 Peptone 6 Dextrose 1 Distilled water to 1 liter.

After 24 hours of incubation at 25 with continuous rotary agitation (280cycles/minute; 2 inch stroke), 5% (vol/vol.) transfers are made to eight250 ml. Erlenmeyer flasks each containing 50 ml. of freshly sterilizedmedium B. After 24 hours of further incubation, using the sameconditions as described above, the steroid, (500 milligrams/ml.) isadded by supplementing each flask with 0.25 ml. of a sterile solutionmg./ml.) of Ila-hydroxy-l9-nor-A -androstadiene-3,17-dione inN,N-dimethylformamide. A total of 200 mg. is fermented. After 48 hoursof further incubation, using identical conditions as described above,the contents of the flasks are pooled, and the broth is extracted threetimes with 200 ml. portions of methyl isobutyl ketone.

I The methyl isobutyl ketone extract is washed with water, dried overanhydrous Na SO and evaporated under vacuum to dryness. The evaporationresidue is chromatographed on a thin layer of Silica Gel GF (Merck) withchloroform containing 10% (by volume) of methanol as the developingsolvent. The major phenolic band as detected by spraying the edge of thethin layer plate with a ferric ferricyanide reagent (Barton, et al.,Nature 170, 249 (1952)) is eluated with a 1:1 (by volume)methanolchloroform. After evaporating the eluate to dryness, the residueis partitioned between equal volumes of chloroform and a 1: l (byvolume) methanol-water mixture. The chloroform phase is replaced twiceand the three chloroform partitionates are combined, dried overanhydrous sodium sulfate, and evaporated to dryness. The evaporationresidue is virtually pure 11a-hydroxy-7-dehydroestrone.

EXAMPLE 14 Following the procedure of Example 13 with the exception thateither 1let-hydroxy-19-nor-A -androstadiene-3, 17-dione or progesteroneis added, the cells of the culture of Cornyebacterium simplex areharvested at the end of 72 hours by centrifugation for ten minutes at2000 G. The supernatant is decanted off and the cells are placed in amortar along with an equal amount by weight of alumina (finely powdered)and treated in a Raytheon magneto-strictive oscillator for 20 minutes.The sonicated mixture is centrifuged for ten minutes at 2000 G to removethe cell debris and alumina.

1lot-hydroxy-l9-A -androstadiene-3,17-dione (1 mg), 2,6-dichlorophenolindophenol (500 g.) or other hydrogen acceptor, such asZ-methylnaphthoquinone and 2.0 ml. of the cell-free ring A dehydrogenasepreparation, described above, are placed in a test tube and brought to avolume of 5.0 ml. with a 0.03 M sodium phosphate buffer. The mixture isallowed to stand for one hour at 30 C. after which it is twice extractedwith 1 ml. of methyl isobutyl ketone. The combined extract ischromatographed on paper using ethylene glycol as the stationary phaseand a mixture of equal volumes of benzene and chloroform as the mobilephase. A spot moving with the same R, (0.10) and exhibiting the samecharacteristic color reactions as the 11a-hydr0xy-7-dehydroestroneobtained in Example 13 is observed.

Similarly, by following the procedures of Example 13 or 14, butsubstituting 11a-hydroxy-19-nor-17u-methyl-7- dehydrotestosterone,1lot-hydroxy-19-nor-17a-ethynyl-7- dehydrotestosterone, and1la-hydroxy-19-nor-7-dehydrotestosterone for the 11a-hydroxy-l9-nor-A-androstadiene-3,l7-dione, the corresponding11a-hydroxy-7-dehydroestrane derivatives are obtained.

9 Similarly, by substituting the following l-dehydrogenatingmicroorganisms for the Corynebacterium simplex in Examples 13 and 14,the same products are formed: Nocwrdia restrictus ATCC 14,887,Pseudam'onas testosteroni ATTC 11,996, Cylindrocarpon radicicola ATTC11,011, and Mycobacterium rhodochrous ATCC 4277.

EXAMPLE 15 2-hydroxy-7-dehydroestrone by growing culture ofCorynebacteriwm simplex (A) Fermentation.-Surface growth from atwo-weekold agar slant of Corynebacterium simplex (ATCC 6946), theslants containing as a nutrient medium (A):

is suspended in 5 m1. of 0.01% aqueous sodium lauryl sulfate solution.One ml. portions of this suspension are used to inoculate four 250 ml.Erlenmeyer flasks, each containing 50 ml. of the following sterilizedmedium (B):

Grams Beef extract 1.5

Yeast extract 3 Peptone 6 Dextrose 1 Distilled water to 1 liter.

After 24 hours of incubation at 25 with continuous rotary agitation (280cycles/minute, 2 inch stroke), 5% (vol/vol.) transfers are made to eight250 ml. Erlenmeyer flasks each containing 50 ml. of freshly sterilizedmedium B. After 24 hours of further incubation, using the sameconditions as described above, the steroid, (500 micrograms/ml.) isadded by supplementing each flask with 0.25 ml. of a sterile solution(100 mg./ml.) of 2ahydroxy 19 nor A androstadiene 3.17 dione inN,N-dimethylformamide. A total of 200 mg. is fermented. After 48 hoursof further incubation, using identical conditions as described above,the contents of the flasks are pooled, and the broth is extracted threetimes with 200 ml. portions of methyl isobutyl ketone. Upon evaporationof the combined extract under vacuum to dryness, crystalline2-hydroxy-7-dehydroestrone is obtained.

EXAMPLE 16 2-hydroxy-7-dehydroestrone by washed cells of Corynebacteriumsimplex Following the procedure of Example 15 with the exception thattestosterone is used in place of 2a-hydroxy- 19-nor-A-androstadiene-3,17-dione, the cells of the culture of Corynebacterz'wmisimplex are harvested at the. end of 72 hours by centrifugation. Thepacked cells are washed three times with a phosphate buffer containing0.005 mole each of KH PQ; and Naz HgpzOq per liter and adjusted to pH7.0. The washed cells are. then suspended in the same phosphate bufferto a volume equal to one-quarter of the volume of the original culture.The substrate, 2a-hydroxy-l9-nor A androstadiene-3,17- dione and thehydrogen acceptor, e.g., Z-methylnaphthoquinone are added as theirsolutions in ethanol to give final concentrations of 100 ,ug./ml. and0.4 mM., respectively, the quantity of ethanol introduced being heldwithin 5% of the total. The reaction mixture is allowed to stand at 30and 4 to 6 hours, after which it is extracted twice with one-quarter ofits volume of methyl isobutyl ketone. The methyl isobutyl ketone extractis Washed twice with water and dried over anhydrous sodium sulfate. Uponevaporating off the solvent to dryness, 2- hydroxy-7-dehydroestrone isobtained as crystalline residue.

10 EXAMPLE 17 2-hydroxy-7-dehydr0estrone by cell-free enzyme preparationfrom Corynebacteriwmsimplex Following the procedure of Example 16, thepacked cells are placed in a mortar along with an equal amount by weightof alumina (finely powdered) and treated in a Raytheon magneto-strictiveoscillator for 20 minutes. The sonicated mixture is centrifuged for tenminutes at 2000 G to remove cell debris and alumina. The substrate, 20chydroxy-19-nor-A -androstadiene-3,l7-dione (1 mg.) and the hydrogenacceptor, e.g., 2-methyl-naphthoquinone (1 mg.) are added to 2 ml. ofthis cell-free ring-A dehydrogenase preparation which has been dilutedto 5 ml. with pH 7.0 phosphate buffer in the same manner as described inExample 16. The. mixture is allowed to stand for one hour at 30. Thecombined extract is chromatographed on paper using ethylene glycol asthe stationary phase and benzene as the mobile phase. A spot moving withthe same Rf (0.15) and exhibiting the same characteristic colorreactions as the 2-hydroxy-7-dehydroestrone obtained in Example. 16 isobserved.

Similarly, by following the procedures of Examples 15 and 16 or 17, butsubstituting ZOL-hYdI'OXY-l9-HOI-l7otmethyl-7-dehydrotestosterone,2a-hydroxy 19' ROI-17ozethynyl-7-dehydrotestosterone, and2a-hydroxy-l9-nor- 7-dehydrotestosterone for the 2a-hydroxy-19-nor-Aandrostadiene-3,l7-dione, the corresponding 2-hydroxy- 7-dehydroestranederivatives are obtained.

EXAMPLE 18 1 1fl-hydroxy-7-dehydroestrone Following the procedure ofExample 13, but substituting 1lfl-hydroxy-l9'-nor-A-androstadiene-3,17-dione, for the. steroid,11B-hydroxy-7-dehydroestrone is obtained.

EXAMPLE 19' fi fl-hydroxy-7-dehydroestrone Following the procedure ofExample 13, but substituting 6fl-hydr0xy-l9 n0r-A"-andr0stadiene-3,l7-di0ne for the steroid, 6B-hydroxy-7-dehydroestroneis obtained.

EXAMPLE 20 16whydroxy-7-dehydroestrone Following the procedure ofExample 13, but substitut ing 16a-hydroxy-19-nor-A-androstadiene-3,17-dione for the steroid, 16a-hydroxy-7-dehydroestroneis obtained.

EXAMPLE 21 14a-hydroxy-7-dehydroestrone Following the procedure ofExample 13, but substituting 14a-hydroxy-19'-nor-A-androstadiene-3,17-dione for the steroid, 14ot-hydroxy-7-dehydroestroneis obtained.

EXAMPLE 22 l5a-hydroxy-7-dehydroestrone Following the procedure ofExample 13, but substituting 15m-hydroxy-19 nor-A-androstadiene-3,l7-dione for the steroid, 15u-hydroxy-7-dehydroestroneis obtained.

EXAMPLE 23 1ZB-hydroxy-7-dehydroestrone Following the procedure ofExample 13, but substituting IZB-hydroxy-l9 nor-A-androstadiene-3,17-dioue for the steroid, 12fi-hydroxy-7-dehydroestroneis obtained.

EXAMPLE 24 1 1'a-hydroxy-7-dehydroestrone 3,1 l-diacetate 11 tate whichseparates is filtered and washed well with water and recrystallized togive 11a-hydroxy-7-dehydroestrone 3,11-diacetate.

Similarly, by following the procedure of Example 24, but substitutingthe following other steroids for the 110:- hydroxy-7-dehydroestrone, thecorresponding diacetates are formed: 2-hydroxy-7-dehydroestrone,6fi-hydroxy-7- dehydroestrone, 15a-hydroxy-7-dehydroestrone and16ahydroxy-7-dehydroestrone.

Moreover, if another acylating agent, such as propionic anhydride andbenzoyl chloride, is substituted for the acetic anhydride in theprocedure of Example 24, the corresponding 3,11-diester is obtained.

EXAMPLE 25 l9-nor-A -androstadiene-3,11,17-trione 11-keto-7-dehydroestrone Following the procedure of Example 13, butsubstituting 19-nor-A "-androstadiene-3,11,17-trione for the steroid,l1-keto-7-dehydr0estrone is obtained.

The invention may otherwise be embodied within the scope of the appendedclaims.

What is claimed is:

1. A compound having the formula:

wherein one X is hydroxy and the remaining Xs are hydrogen, or where twoXs are connected to the same car- 12 bon, these two Xs can be oxo; Y ishydroxy, Z is hydrogen, methyl or ethynyl, or together Y and Z is 0x0.

2. A compound having the formula:

wherein one R is R'O and the remaining Rs are hydrogen, or where two Rsare connected to the same carbon atom, these two Rs can be 0x0; R ishydrogen or the acyl radical of a hydrocarbon carboxylic acid of lessthan twelve carbon atoms; Y is hydroxy, Z is hydrogen, methyl orethynyl, or together Y and Z is 0x0.

3. A compound having the formula:

wherein one X is hydroxy and the remaining Xs are hydrogen.

4. The compound of claim 1 having the name hydroxy-19-nor-A-androstadiene-3,17-dione.

5. The compound of claim 1 having the name l9-nor- A -androstadiene-3,11,17-trione.

6. The compound of claim 3 having the name Ila-hydroxy-7-dehydroestrone.

References Cited UNITED STATES PATENTS 6/1967 Kriiger 260--397.4 9/1969Marshall 260239.55

LEWIS GOTTS, Primary Examiner E. G. LOVE, Assistant Examiner US. Cl.X.R.

wgg UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Pat t N 3 517036 Dated June 23 1970 Inventofls) Patrick A. Diassi It is certifiedthat error appears in the above-identified patent and that said LettersPatent are hereby corrected as shown below:

filolumn 2, line 56, "product" should read-products-. Column 5 line 46,"concial" should read--conical--. Column 3, line 58, delete--lO%--.Column 3, line 60, "milligrams ml. should read--milligrams/ml. Column 5line 66, "minutes" should read-minute--. Column 7, line 3, "Seizt"should read--Seitz--. Column 7, line 32, "l6-dhydroxy" shouldread-l6dhydroxy--. Column 9, line 5, "ATTC" should read-ATCC-. Column 9,line 40, "3. 17" should rea.d3 l7-. Column 9, line 69, "and" shouldread-'---for. Claim 1 in the formula should be:

si ssd and sealed this 30th day of March 1971.

(SEAL) Attsst:

EDWARD M.FL1.. CHER,JH. WILLIAM E. SCHUYLER, JR.

Attesting OLicer Commissioner of Patents

